The organization of 3' splice-site sequences in mammalian introns.

A model pre-mRNA substrate was used to carry out a detailed investigation of the functional organization of sequences at the 3' end of mammalian introns. This analysis revealed a difference in the sequence requirements for the first and second steps of the splicing reaction (lariat formation and exon ligation, respectively). Maximal efficiencies of lariat formation require a pyrimidine stretch directly adjacent to the branch site. In addition, efficient lariat formation can be specified in at least two distinct ways, one that requires the AG dinucleotide at the 3' splice junction, and the other that does not: If the pyrimidine stretch is short (14 nucleotides), an adjacent AG is essential; in contrast, the AG is not required in the presence of a long pyrimidine stretch (26 nucleotides). In a pre-mRNA containing a long pyrimidine stretch, efficient lariat formation is observed when the branch site is located greater than 100 nucleotides upstream from the AG or when the AG is preceded by a purine stretch. Although splicing usually takes place at the first AG downstream from the branch site, both distance and sequence play roles in the efficiency of this reaction.